Mycobacterium marinum is the causative agent of localized lesions in the extremities, commonly called swimming pool or aquarium granuloma. Swimming pool granuloma is relatively common in professions involving almost any water source, including aquarium maintenance, fishing and diving. Currently, infections occur at a frequency of nearly 200 per year in the U.S. alone and are thought to be on the rise. Improved methods for disease prevention are a high priority, since antibiotic therapy usually requires multiple drugs and from four to fourteen months before full recovery. M. marinum replicates primarily intracellularly in macrophages during disease in humans. Thus, M. marinum must enter a eukaryotic cell in order to replicate and the genes involved in entry should be critical for production of disease. Our previous studies have resulted in the discovery of two key aspects of the ability of M. marinum to invade host cells. First, we have found that M. marinum enters host cells at up to 100 fold higher levels than the non-pathogenic mycobacterial strain M. smegmatis. This entry mechanism appears to enhance the ability of M. marinum to survive intracellularly. Second, the ability of M. marinum to enter host cells is regulated by growth conditions. We have utilized these data to develop novel strategies for isolation of the genes required for entry of M. marinum into host cells. The close genetic relationship of M. marinum to other mycobacterial species such as M. avium and particularly M. tuberculosis, suggests that the genes identified may play a similar role in other pathogenic mycobacteria. Our hypothesis is that the ability to enter host cells is important for virulence of mycobacteria. The specific aims of the current proposal are: 1) isolate and characterize mycobacterial genes involved in entry and 2) determine the involvement of these genes in virulence in mice. Through an examination of the mechanisms of entry and the factors that regulate it, we hope to further our understanding of how mycobacteria cause disease as well as provide insight into novel methods for their prevention.